Asymmetric distribution of the Na+/H+ antiporter in the renal proximal tubule epithelial cell.
Asymmetric distribution of the Na+/H+ antiporter in the renal proximal tubule epithelial cell.
复制标题
Na /H 逆向转运蛋白在肾近曲小管上皮细胞中的不对称分布。
DOI：
10.1016/s0021-9258(17)43943-3
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发表时间：
1983
期刊：
影响因子：
--
通讯作者：
D. Warnock
中科院分区：
文献类型：
--
作者：
H. Ives;V. Yee;D. Warnock
用差速离心和密度梯度离心法研究了Na ~+/H ~+逆向转运蛋白在肾近曲小管细胞的亚细胞分布。在蔗糖密度梯度中同时测定基底外侧膜（Na+/K+）-ATP酶）、刷状缘膜（麦芽糖酶）和各种细胞内细胞器（NADPH细胞色素c还原酶、硫胺素焦磷酸酶、酸性磷酸酶和琥珀酸细胞色素c还原酶）的酶标记物。通过该技术，基底外侧膜（中位rho = 1.150）与刷状缘膜（中位rho = 1.165）分离良好。其他细胞器的标记物有中间或双峰分布。为了确定Na+/H+反向转运蛋白的细胞位置，使用吖啶橙子在蔗糖密度梯度级分中测定预先形成的pH梯度的Na+依赖性崩溃。Na+/H+逆向转运蛋白活性与刷状缘膜组分的分布一致，基底外侧膜组分的活性低于刷状缘组分的5%。为了确定在所有细胞级分中是否都可能检测到反向转运蛋白活性，将尼日利亚菌素加入到每个级分中，并用吖啶橙子测定K+/H+交换。活性存在于所有蔗糖密度梯度级分中。此外，有没有改变，在刷状缘膜与细胞溶胶或基底膜混合后测量的Na+/H+交换活性，表明既不抑制剂，也不激活的Na+/H+逆向转运蛋白存在于任何细胞组分。这些对照证实了基底外侧膜不存在Na+/H+反向转运蛋白活性的发现。刷状缘膜中存在Na+/H+反向转运蛋白而基底外侧膜中不存在，这与其在H+从血液向肾近端小管管腔的矢量转运中发挥重要作用一致。
The subcellular distribution of the Na+/H+ antiporter in renal proximal tubule cells was studied with differential and density gradient centrifugation. Enzyme markers for basolateral membranes [Na+/K+)-ATPase), brush border membranes (maltase), and a variety of intracellular organelles (NADPH cytochrome c reductase, thiamine pyrophosphatase, acid phosphatase, and succinate cytochrome c reductase) were simultaneously assayed in sucrose density gradients. Basolateral membranes (median rho = 1.150) were well separated from brush border membranes (median rho = 1.165) by this technique. Markers for other cellular organelles had intermediate or bimodal distributions. To determine the cellular location of the Na+/H+ antiporter, Na+-dependent collapse of preformed pH gradients was assayed in the sucrose density gradient fractions using acridine orange. Na+/H+ antiporter activity paralleled the distribution of the brush border membrane fractions; activity in the peak basolateral membrane fraction was less than 5% of that in the peak brush border fraction. To determine whether antiporter activity was potentially detectable in all cell fractions, nigericin was added to each fraction and K+/H+ exchange was assayed with acridine orange. Activity was present in all sucrose density gradient fractions. In addition, there was no alteration in Na+/H+ exchange activity measured in brush border membranes after mixing with cell sol or basolateral membranes, showing that neither inhibitors nor activators of the Na+/H+ antiporter were present in any of the cell fractions. These controls confirmed the finding that Na+/H+ antiporter activity was absent from basolateral membranes. The presence of the Na+/H+ antiporter in brush border membranes and its absence from basolateral membranes is consistent with its playing an important role in the vectorial transport of H+ from blood to tubular lumen in the renal proximal tubule.